CAJ | 학술논문

目的探讨miR-720在急性髓系白血病(AML)患者中的表达水平、调控机制及其与白血病肿瘤生物学行为的关系.方法采用荧光定量PCR检测38例AML患者和20名正常对照骨髓细胞miR-720表达水平;采用焦磷酸测序法定量检测AML患者和正常对照miR-720启动子甲基化水平;构建慢病毒介导的miR-720过表达kasumi-1细胞,采用流式细胞术、WST-1法、集落形成实验、Transwell细胞迁移实验及Western blot法分别检测miR-720对kasumi-1细胞增殖、凋亡、细胞周期、集落生成、迁移及P53介导的细胞凋亡通路的影响.采用重亚硫酸盐测序法检测地西他滨处理前后kasumi-1细胞miR-720甲基化水平,采用荧光定量PCR检测地西他滨处理后kasumi-1细胞miR-720表达水平.结果与正常对照相比,AML患者miR-720表达水平明显降低(0.69±0.09对3.00±0.46,P＜0.01),且miR-720启动子区域呈现显著高水平的DNA甲基化[(75.56±2.35)％对(47.65±2.78)％,P＜0.01].miR-720过表达的kasumi-1细胞自身凋亡率明显增高(P=0.017),对细胞毒药物依托泊苷的凋亡敏感性也明显增加(P=0.004),细胞增殖明显受抑(P＜0.01),细胞集落形成能力减弱(P=0.005),细胞周期阻滞于G1/G0期,细胞迁移能力下降.miR-720的过表达显著诱导kasumi-1细胞P53凋亡相关蛋白的表达包括P53、Bax以及诱导NF-κB信号转导通路的激活.地西他滨处理后的kasumi-1细胞miR-720表达水平较处理前明显增高,而启动子甲基化水平明显降低.结论 AML患者骨髓细胞miR-720表达水平明显降低,DNA甲基化介导的miR-720表达抑制有助于白血病生物学特征的维持.
목적 탐토miR-720재급성수계백혈병(AML)환자중적표체수평、조공궤제급기여백혈병종류생물학행위적관계.방법 채용형광정량PCR검측38례AML환자화20명정상대조골수세포miR-720표체수평;채용초린산측서법정량검측AML환자화정상대조miR-720계동자갑기화수평;구건만병독개도적miR-720과표체kasumi-1세포,채용류식세포술、WST-1법、집락형성실험、Transwell세포천이실험급Western blot법분별검측miR-720대kasumi-1세포증식、조망、세포주기、집락생성、천이급P53개도적세포조망통로적영향.채용중아류산염측서법검측지서타빈처리전후kasumi-1세포miR-720갑기화수평,채용형광정량PCR검측지서타빈처리후kasumi-1세포miR-720표체수평.결과 여정상대조상비,AML환자miR-720표체수평명현강저(0.69±0.09대3.00±0.46,P＜0.01),차miR-720계동자구역정현현저고수평적DNA갑기화[(75.56±2.35)％대(47.65±2.78)％,P＜0.01].miR-720과표체적kasumi-1세포자신조망솔명현증고(P=0.017),대세포독약물의탁박감적조망민감성야명현증가(P=0.004),세포증식명현수억(P＜0.01),세포집락형성능력감약(P=0.005),세포주기조체우G1/G0기,세포천이능력하강.miR-720적과표체현저유도kasumi-1세포P53조망상관단백적표체포괄P53、Bax이급유도NF-κB신호전도통로적격활.지서타빈처리후적kasumi-1세포miR-720표체수평교처리전명현증고,이계동자갑기화수평명현강저.결론 AML환자골수세포miR-720표체수평명현강저,DNA갑기화개도적miR-720표체억제유조우백혈병생물학특정적유지.
Objective To investigate the expression level and regulation mechanism of miR-720 as well as the association of miR-720 expression with leukemia biological characteristics.Methods Expression and promoter methylation of miR-720 were determined by quantitive PCR and pyrosequencing in 38 patients with AML and 20 normal controls.Lentivirous-mediated miR-702 overexpression was constructed in AML cell line kasumi-1.The cell proliferation,apoptosis,cycle,colony formation,migration and P53-mediated apoptosis pathway were determined.Results AML patients showed significantly lower miR-720 expression compared with normal controls (0.69 ± 0.09 vs 3.00 ± 0.46,P＜0.01); The methylation level of miR-720 promoter region in AML patients were significantly higher than normal controls [(75.56± 2.35)％ vs (47.65 ± 2.78)％,P＜0.01].miR-720 overexpression in kasumi-1 cells induced significantly increased cell apoptosis (P=0.017),elevated apoptosis sensitivity to etoposide (P=0.004),and reduced cell proliferation (P＜0.01).miR-720 overexpression also induced reduced colony formation (P=0.005),cell cycle arrest in G1/G0 phase and decreased migration ability in kasumi-1 cells.In addition,overexpression of miR-720 significantly induced increased cell apoptosis-related proteins including P53 and Bax,and activation of NF-κB signal transduction pathway.After kasumi-1 cells were treated with 1 uM decitabine for 48 hours,miR-720 promoter methylation reduced significantly,and miR-720 expression significantly increased.Conclusion The expression of miR-720 in AML patients reduced significantly,and DNA methylation-mediated epigenetic silencing of miR-720 contributed to maintain the malignant characteristics of AML.